bFGF诱导的增殖人晶体上皮细胞内Ca～(2+)调控的初步研究
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摘要
白内障为世界上致盲疾病之首，但目前为之有效的治疗手段仍然是手术治疗。但术后，很多患者出现了晶体后囊混浊，即后发性白内障(PCO)，导致视力再度下降。虽然手术或激光后囊切开是一较为有效的治疗方法，但后囊膜的开通可导致视网膜脱离、黄斑水肿等严重的并发症，为患者再次造成痛苦和损失。因此，如何预防和药物治疗白内障及后发障是目前眼科工作者亟待解决的迫切问题。
已证实，术后晶体上皮细胞(LEC)的残留和增殖是后囊膜混浊的形成基础。生长因子(GF)在此过程中发挥着极为重要的作用，是促进术后LEC增殖，分化，移行引起后发障的主要因素，其中bFGF的作用尤为突出。
研究发现：晶体细胞内钙也有着明显地促晶体上皮细胞生长作用；许多生长因子如EGF、PDGF、bFGF对某些细胞内Ca~(2+)有一定的调节作用；而钙动员对生长因子也有一定的调节作用，在一些实验中发现细胞内Ca~(2+)的释放也可增加小脑颗粒神经元内bFGF的表达。二者在细胞内信号转导和细胞增殖、分化过程中存在着相互作用。
基于以上背景我们作出大胆推测：在晶体上皮细胞中，GF对Ca~(2+)也存在着一定的调节作用，这种调节引起的钙动员可能也参与了GF促细胞增殖过程，并发挥着协同的作用。因此，找出GF与Ca~(2+)的关系，发现它们的作用方式，特异性阻断两者在晶体上皮细胞增殖过程中的相互作用，将为白内障尤其是后发障的预防和治疗提供更为广阔的思路。
为证实所作推测，本实验从晶体上皮细胞内钙库通道三磷酸肌醇受体(IP3R)和Ryanodine受体(RyR)入手，首先判定它们在人晶体上皮细胞(HLEC)内的表达，推断它们在HLEC内Ca~(2+)调节中的作用，然后用bFGF诱导HLEC增殖，检测bFGF对HLEC内Ca~(2+)、IP3R、RyR的作用，最后通过药理反应推断bFGF对HLEC内Ca~(2+)的作用途径，
——
为进一步药物阻断GF与Ca卜之间的相互作用从而抑制晶体上皮细胞的
增殖提供了理论基础。
方 法
第一部分：
卫、人晶体上皮细胞系传代培养
2、取第三代细胞，TRIZOI试剂舰 HLEC内和各受体亚型阳性对
照组织内总RNA（阳M照组织选自足月死婴），RT－PCR方法检测
HuC内 IP3RI，11，Ill和 RyRI，11，Ill亚型 m删的趟。各弓；物根
据NCBI公布的人类以上各受体mRNA序列触用软件Primer3所得。
实验均重复四次，Kodak ID digital science凝胶图像撕软件撕凝胶
电泳结果，计算目的基因mRNA的相对含量，作两样本均数差异的t
检验。
3、取同代细胞传代与30mm培养皿中，在激光扫描共聚焦显微镜
下（LSCM）向各组内分别加入一定浓度乙酚胆碱、阿托品、咖啡因、
yanodine、普鲁卡因溶液（各物质均溶解于不含有 C扩”，Mgl”的 PBS
液中，在 37 C下孵育 20分钟）通过观测相对荧光密度实时观察细胞内
Caz”变化。
第二部分：
1、w＊F诱导＊LE C体外增殖及测定：选取第三代培养细胞，以
浓度为 10帅1加入 96孔培养板中，每孔加 100…含 2％FSC的
DMEM，与孵箱内培养2－3小时，待细胞贴壁。之后向各孔分别加培
养液稀释后的＊＊＊F10O卜 使其终鹏分别为且n咖互，二ong／ml，100
n咖I，阴性对照每孔加 DMEM 100…。各浓度梯度及对照组各 3孔。
第三珊行MTf测定。各组比色阶t检验。
2、RT－PCR检测 10n卵lbFGF 作用后的HuC内IP3R和RyR
各亚型mRNA表达变化
3、LSCM下检测 Inghl，10ng／ml，100 ＂g／ffil bFGF作用后第三天
HLEC内静息Ca卜浓度变化。
第三部分：
取第三代细胞，在激光扫描共聚焦显微镜下向各组内分别加入
2
————
10nghl bFGF，10mM咖啡因；10mM Ryanodine、50InM普鲁卡因溶
液，05mM Genistein，通过MI细胞内相对荧掷度实时观察细胞内 Cah
变化。
结 果
第一部分：
1、传代后的细胞24小时后几乎完全贴壁，细胞呈多形性，第三代
细胞出现大量梭状或长条状。细胞分裂较快，36小时即开始增殖。在
接种密度为 105细w毫升时，一周内即可连接成片。
2、HLEC存在IP3RI、Ill型和RyRI、Ill型m聊表达，且都以＊
型表达为主。P（1oR）＜0＊5；P（RyR）＜0．0】
3、10pM Ach在细胞外液含Ca卜，Mg卜的翩下，可腿G；起HLEC
内［Ca卜］的升高，IPM阿托品即可基本阻断 Ach的作用。在细胞外
液无 Cah，Mgl“含 lmM EGI？A时，Ach仍能较迅速引起［Ca卜］的升
高，但较前者持续时间短；1（）mM Caffeine可腿引起细胞内【Ca卜］
的逐步升高，幅度较Ach小，但持续时间较长。另一方面，追加10pM Ach，
并未再次弓！起［Caf”］的升高；50pM Ry。flodillC使细胞内钙［C。‘”］
浓髓慢升高，持续时间舰长，幅度与Caffeine相似；50PM Procaine
可显著抑制Ryano山ne的活性。
第二部分：
l、MTI’测定结果bFGI’呈浓度懈性诱导HLEC的增殖，其中
10fig／ml的作用最为显著。P刃＊
2、bFGF诱导后 HLEC实鞭和对照组内仍然可见 IP3RI、Ill，RyRI、
Cataract is a major disease leading to blindness and surgery is still the most available therapy method for it. While posterior capsule opacification (PCO) occurs in approximately 18-48% cases of adults and even almost 100% of kids after planned extracapsular cataract extraction (ECCE). Although Laser Nd:YAG capsulotomy or secondary surgery can help restore visual acuity, the complications such as retinal detachment and macular edema will bring the patients more troubles. Therefore, finding some other ways to prevent the occurrence of the cataract and PCO or cure the disease is an emergency problem.
As established, PCO is due to the proliferation, differentiation, and migration of the residual cells over the lens capsule equator and under the anterior capsule. Growth factors such as basic fibroblast growth factor (bFGF) play a vital role in this process.
On the other hand, we have known the imbalance of intracellular calcium ion concentration especially the increase of calcium ion concentration in lens epithelial cells is one of the causes of cataract and PCO. In almost all of kinds of cataract, the calcium ion level is higher than normal. Many researches have done on drug usage to inhibit the calcium increase in order to prevent cataract.
In early researches, it is found that many growth factors including epidermal growth factor, platelet derived growth factor and basic fibroblast growth factor could regulate the intracellular calcium in various patterns. For example: bFGF increases functional L-type Ca2+ channels on fetal rat hippocampal neurons; in human retinal pigment epithelial cells, EGF, PDGF, FGF can all induce a transient calcium increase; both of FGF and PDGF have
been shown to induce calcium release from intracellular stores in a range of different cell types.
Calcium mobilisation also contributes to lens epithelial cells growth obviously. In some studies, intracellular calcium release can increase bFGF expression in cerebellar granule neurons. Calcium and growth factors have interactions in intracelluar signal transduction, cell proliferation and differentiation.
In view of the backgroud above, we guess grwoth factor (GF) can also regulate the intracellular calcium in lens epithelial cells, and the two play some important role together in lens epithelial cells proliferation, migragation, differentiation. Therefore to find their relationship, detect their action pattern and block their interaction specifically will provide a new foregroud for cataract and PCO prevention or treatment.
In my reaserch, I focued on the function of inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR), which present the two intracellular calcium pool in LEG during the calcium signal transduction especially in duced by bFGF. First, to determinate the existence of IP3R and RyR expression and assess their function during calcium ion concentration modulation in cultured human lens epithelial cells. Then try to find out the effects of bFGF (basic fibroblast growth factor) on the Ca2+ concentration and the expression of IP3R and RyR. Finally, to investigate the pathway of calcium regulated by bFGF through pharmacological reaction. What we have done will provide theory basis for further drug application in order to block GF and Ca2+ interaction for LEG proliferation inhibition.
The reaserch is divided into three parts:
Parti:
The Coexistence of IP3R and RyanodineR in Human Lens Epithelial Cells
Objective: To determinate the existence of the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR), identify their isoforms and assess their functions during calcium ion concentration modulation in cultured human lens epithelial cells. Methods: Human lens epithelial cells were subcultured, the 3rd passage cells
were chosen as object when they become confluent. Then the total cellular RNA was extracted with TRIzol from the cells and positive control tissues (from term dead fetus). The expression of mRNA of IP3R and RyR isoforms I, II, III was analyzed by
已证实，术后晶体上皮细胞(LEC)的残留和增殖是后囊膜混浊的形成基础。生长因子(GF)在此过程中发挥着极为重要的作用，是促进术后LEC增殖，分化，移行引起后发障的主要因素，其中bFGF的作用尤为突出。
研究发现：晶体细胞内钙也有着明显地促晶体上皮细胞生长作用；许多生长因子如EGF、PDGF、bFGF对某些细胞内Ca~(2+)有一定的调节作用；而钙动员对生长因子也有一定的调节作用，在一些实验中发现细胞内Ca~(2+)的释放也可增加小脑颗粒神经元内bFGF的表达。二者在细胞内信号转导和细胞增殖、分化过程中存在着相互作用。
基于以上背景我们作出大胆推测：在晶体上皮细胞中，GF对Ca~(2+)也存在着一定的调节作用，这种调节引起的钙动员可能也参与了GF促细胞增殖过程，并发挥着协同的作用。因此，找出GF与Ca~(2+)的关系，发现它们的作用方式，特异性阻断两者在晶体上皮细胞增殖过程中的相互作用，将为白内障尤其是后发障的预防和治疗提供更为广阔的思路。
为证实所作推测，本实验从晶体上皮细胞内钙库通道三磷酸肌醇受体(IP3R)和Ryanodine受体(RyR)入手，首先判定它们在人晶体上皮细胞(HLEC)内的表达，推断它们在HLEC内Ca~(2+)调节中的作用，然后用bFGF诱导HLEC增殖，检测bFGF对HLEC内Ca~(2+)、IP3R、RyR的作用，最后通过药理反应推断bFGF对HLEC内Ca~(2+)的作用途径，
——
为进一步药物阻断GF与Ca卜之间的相互作用从而抑制晶体上皮细胞的
增殖提供了理论基础。
方 法
第一部分：
卫、人晶体上皮细胞系传代培养
2、取第三代细胞，TRIZOI试剂舰 HLEC内和各受体亚型阳性对
照组织内总RNA（阳M照组织选自足月死婴），RT－PCR方法检测
HuC内 IP3RI，11，Ill和 RyRI，11，Ill亚型 m删的趟。各弓；物根
据NCBI公布的人类以上各受体mRNA序列触用软件Primer3所得。
实验均重复四次，Kodak ID digital science凝胶图像撕软件撕凝胶
电泳结果，计算目的基因mRNA的相对含量，作两样本均数差异的t
检验。
3、取同代细胞传代与30mm培养皿中，在激光扫描共聚焦显微镜
下（LSCM）向各组内分别加入一定浓度乙酚胆碱、阿托品、咖啡因、
yanodine、普鲁卡因溶液（各物质均溶解于不含有 C扩”，Mgl”的 PBS
液中，在 37 C下孵育 20分钟）通过观测相对荧光密度实时观察细胞内
Caz”变化。
第二部分：
1、w＊F诱导＊LE C体外增殖及测定：选取第三代培养细胞，以
浓度为 10帅1加入 96孔培养板中，每孔加 100…含 2％FSC的
DMEM，与孵箱内培养2－3小时，待细胞贴壁。之后向各孔分别加培
养液稀释后的＊＊＊F10O卜 使其终鹏分别为且n咖互，二ong／ml，100
n咖I，阴性对照每孔加 DMEM 100…。各浓度梯度及对照组各 3孔。
第三珊行MTf测定。各组比色阶t检验。
2、RT－PCR检测 10n卵lbFGF 作用后的HuC内IP3R和RyR
各亚型mRNA表达变化
3、LSCM下检测 Inghl，10ng／ml，100 ＂g／ffil bFGF作用后第三天
HLEC内静息Ca卜浓度变化。
第三部分：
取第三代细胞，在激光扫描共聚焦显微镜下向各组内分别加入
2
————
10nghl bFGF，10mM咖啡因；10mM Ryanodine、50InM普鲁卡因溶
液，05mM Genistein，通过MI细胞内相对荧掷度实时观察细胞内 Cah
变化。
结 果
第一部分：
1、传代后的细胞24小时后几乎完全贴壁，细胞呈多形性，第三代
细胞出现大量梭状或长条状。细胞分裂较快，36小时即开始增殖。在
接种密度为 105细w毫升时，一周内即可连接成片。
2、HLEC存在IP3RI、Ill型和RyRI、Ill型m聊表达，且都以＊
型表达为主。P（1oR）＜0＊5；P（RyR）＜0．0】
3、10pM Ach在细胞外液含Ca卜，Mg卜的翩下，可腿G；起HLEC
内［Ca卜］的升高，IPM阿托品即可基本阻断 Ach的作用。在细胞外
液无 Cah，Mgl“含 lmM EGI？A时，Ach仍能较迅速引起［Ca卜］的升
高，但较前者持续时间短；1（）mM Caffeine可腿引起细胞内【Ca卜］
的逐步升高，幅度较Ach小，但持续时间较长。另一方面，追加10pM Ach，
并未再次弓！起［Caf”］的升高；50pM Ry。flodillC使细胞内钙［C。‘”］
浓髓慢升高，持续时间舰长，幅度与Caffeine相似；50PM Procaine
可显著抑制Ryano山ne的活性。
第二部分：
l、MTI’测定结果bFGI’呈浓度懈性诱导HLEC的增殖，其中
10fig／ml的作用最为显著。P刃＊
2、bFGF诱导后 HLEC实鞭和对照组内仍然可见 IP3RI、Ill，RyRI、
Cataract is a major disease leading to blindness and surgery is still the most available therapy method for it. While posterior capsule opacification (PCO) occurs in approximately 18-48% cases of adults and even almost 100% of kids after planned extracapsular cataract extraction (ECCE). Although Laser Nd:YAG capsulotomy or secondary surgery can help restore visual acuity, the complications such as retinal detachment and macular edema will bring the patients more troubles. Therefore, finding some other ways to prevent the occurrence of the cataract and PCO or cure the disease is an emergency problem.
As established, PCO is due to the proliferation, differentiation, and migration of the residual cells over the lens capsule equator and under the anterior capsule. Growth factors such as basic fibroblast growth factor (bFGF) play a vital role in this process.
On the other hand, we have known the imbalance of intracellular calcium ion concentration especially the increase of calcium ion concentration in lens epithelial cells is one of the causes of cataract and PCO. In almost all of kinds of cataract, the calcium ion level is higher than normal. Many researches have done on drug usage to inhibit the calcium increase in order to prevent cataract.
In early researches, it is found that many growth factors including epidermal growth factor, platelet derived growth factor and basic fibroblast growth factor could regulate the intracellular calcium in various patterns. For example: bFGF increases functional L-type Ca2+ channels on fetal rat hippocampal neurons; in human retinal pigment epithelial cells, EGF, PDGF, FGF can all induce a transient calcium increase; both of FGF and PDGF have
been shown to induce calcium release from intracellular stores in a range of different cell types.
Calcium mobilisation also contributes to lens epithelial cells growth obviously. In some studies, intracellular calcium release can increase bFGF expression in cerebellar granule neurons. Calcium and growth factors have interactions in intracelluar signal transduction, cell proliferation and differentiation.
In view of the backgroud above, we guess grwoth factor (GF) can also regulate the intracellular calcium in lens epithelial cells, and the two play some important role together in lens epithelial cells proliferation, migragation, differentiation. Therefore to find their relationship, detect their action pattern and block their interaction specifically will provide a new foregroud for cataract and PCO prevention or treatment.
In my reaserch, I focued on the function of inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR), which present the two intracellular calcium pool in LEG during the calcium signal transduction especially in duced by bFGF. First, to determinate the existence of IP3R and RyR expression and assess their function during calcium ion concentration modulation in cultured human lens epithelial cells. Then try to find out the effects of bFGF (basic fibroblast growth factor) on the Ca2+ concentration and the expression of IP3R and RyR. Finally, to investigate the pathway of calcium regulated by bFGF through pharmacological reaction. What we have done will provide theory basis for further drug application in order to block GF and Ca2+ interaction for LEG proliferation inhibition.
The reaserch is divided into three parts:
Parti:
The Coexistence of IP3R and RyanodineR in Human Lens Epithelial Cells
Objective: To determinate the existence of the inositol 1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR), identify their isoforms and assess their functions during calcium ion concentration modulation in cultured human lens epithelial cells. Methods: Human lens epithelial cells were subcultured, the 3rd passage cells
were chosen as object when they become confluent. Then the total cellular RNA was extracted with TRIzol from the cells and positive control tissues (from term dead fetus). The expression of mRNA of IP3R and RyR isoforms I, II, III was analyzed by
引文
1． Reddy VN et al.Effects of growth factors on proliferation and differentiation in human lens epithelial cells in early subculture.Invest Ophthalmol Vis Sci.1995 Oct;36(11) :2304-12．
2． Reddan JR e tal.Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.Invest Ophthalmol Vis Sci.1995 Feb;36(2) :509-13．
3． 申图形超，姚克，等。BFGF对人晶状体上皮细胞增殖的影响。眼 科新进展，2000，20：118-120
4． 刘晓莉 眼科研究1996 Vol 14 Nol 13-15
5． Thmos R.Shearer Shearer,Mol Vis 1997,3,8
6． Kenneth R.Hightower Cun.Eye Res.14:71-78,1995
7． Li WC,Kuszak JK,Exp Eye Res.1995,61:91-98
8． 王凤翔 何守致 中华眼科1998。Vol 34 No．5 352-354
9． Shoji Kuriyama,Teruyo Ohuchi,Nagahisa Yoshimura,et al.Growth Factor-induced Cytosolic Calcium Ion Transients in Cultured Human Retinal Pigment Epithelial Cells.Invest Ophthalmol Vis Sci 32:2882,1991
10． Shitaka Y,Matsuki N,Saito H,et al.Basic fibroblast growth factor increases functional L-type Ca2+ channels in fetal rat hippocampal neurons:implications for neurite morphogenesis in vitro.J Neurosci 1996 Oct 15;16(20) :6476-89
11． Emmanuel Samain;Helene Bouillier;Stephanie Miserey.Extracellular Signal-Regulated Kinase Pathway Is Involved in Basic Fibroblast Growth Factor Effect on Angiotensin II-Induced Ca2+ Transient in Vascular Smooth Muscle Cell From Wistar-Kyoto and Spontaneously Hypertensive Rats.Hypertension.2000;35:61
12． Duncan G,Wormstone IM,Liu CS,et al.Thapsigargin-coated intraocular lenses inhibit human lens cell growth.Nature Med 1997 Sep;3(9) :1026-8．
13． Duncan G,Williams MR,Riach RA.Calcium,cell signallyng and cataract.Prog Ret Eye Res 1994;13:623-52
14． Sanderson MJ,et al.Mechanisms and function of intracellular calcium
signaling.Nature,1993,361:315-325
15． Ferris CD,Huganir RL.Purified inositol 1,4,5-trisphosphate receptor mediated calcium flux in reconstituted lipid vesicles.Nature 1989 342,87-89
16． John L.Sutko,Judith A.Airey,The Pharmacology of Ryanodine and Related Compounds ?Pharmological Reviews 1997 March Vol.49,Issue 1,53-98,
17． Riccardo Zucchi,Simonetta Ronca-Testoni.The Sarcoplasmic Reticulum Ca2+ Channel/Ryanodine Receptor:Modulation by Endogenous Effectors,Drugs and Disease States.Pharmological Reviews 1997 March Vol.49,Issue 1,1-52
18． 张川星，吴本介．钙荧光探剂的研究及其在生命科学中的应用．生 理科学进展，1996，27：1．
2． Reddan JR e tal.Establishment of epithelial lines from cryopreserved lenses and capsule-epithelial preparations.Invest Ophthalmol Vis Sci.1995 Feb;36(2) :509-13．
3． 申图形超，姚克，等。BFGF对人晶状体上皮细胞增殖的影响。眼 科新进展，2000，20：118-120
4． 刘晓莉 眼科研究1996 Vol 14 Nol 13-15
5． Thmos R.Shearer Shearer,Mol Vis 1997,3,8
6． Kenneth R.Hightower Cun.Eye Res.14:71-78,1995
7． Li WC,Kuszak JK,Exp Eye Res.1995,61:91-98
8． 王凤翔 何守致 中华眼科1998。Vol 34 No．5 352-354
9． Shoji Kuriyama,Teruyo Ohuchi,Nagahisa Yoshimura,et al.Growth Factor-induced Cytosolic Calcium Ion Transients in Cultured Human Retinal Pigment Epithelial Cells.Invest Ophthalmol Vis Sci 32:2882,1991
10． Shitaka Y,Matsuki N,Saito H,et al.Basic fibroblast growth factor increases functional L-type Ca2+ channels in fetal rat hippocampal neurons:implications for neurite morphogenesis in vitro.J Neurosci 1996 Oct 15;16(20) :6476-89
11． Emmanuel Samain;Helene Bouillier;Stephanie Miserey.Extracellular Signal-Regulated Kinase Pathway Is Involved in Basic Fibroblast Growth Factor Effect on Angiotensin II-Induced Ca2+ Transient in Vascular Smooth Muscle Cell From Wistar-Kyoto and Spontaneously Hypertensive Rats.Hypertension.2000;35:61
12． Duncan G,Wormstone IM,Liu CS,et al.Thapsigargin-coated intraocular lenses inhibit human lens cell growth.Nature Med 1997 Sep;3(9) :1026-8．
13． Duncan G,Williams MR,Riach RA.Calcium,cell signallyng and cataract.Prog Ret Eye Res 1994;13:623-52
14． Sanderson MJ,et al.Mechanisms and function of intracellular calcium
signaling.Nature,1993,361:315-325
15． Ferris CD,Huganir RL.Purified inositol 1,4,5-trisphosphate receptor mediated calcium flux in reconstituted lipid vesicles.Nature 1989 342,87-89
16． John L.Sutko,Judith A.Airey,The Pharmacology of Ryanodine and Related Compounds ?Pharmological Reviews 1997 March Vol.49,Issue 1,53-98,
17． Riccardo Zucchi,Simonetta Ronca-Testoni.The Sarcoplasmic Reticulum Ca2+ Channel/Ryanodine Receptor:Modulation by Endogenous Effectors,Drugs and Disease States.Pharmological Reviews 1997 March Vol.49,Issue 1,1-52
18． 张川星，吴本介．钙荧光探剂的研究及其在生命科学中的应用．生 理科学进展，1996，27：1．